F1000Research 2021, 10:645 Last updated: 27 JUL 2021 RESEARCH ARTICLE The discovery of human Plasmodium among domestic animals in West Sumba and Fakfak, Indonesia [version 1; peer review: awaiting peer review] Munirah Munirah 1, Sitti Wahyuni2, Isra Wahid 2, Firdaus Hamid3 1Doctoral Study Program, Faculty of Medicine, Hasanuddin University, Makassar. Jln. Perintis Kemerdekaan 10 Tamalanrea, Makassar, South Sulawesi, 90245, Indonesia 2Department of Parasitology, Faculty of Medicine, Hasanuddin University, Makassar. Jln. Perintis Kemerdekaan 10 Tamalanrea, Makassar, South Sulawesi, 90245, Indonesia 3Department of Microbiology, Faculty of Medicine, Hasanuddin University, Makassar. Jln. Perintis Kemerdekaan 10 Tamalanrea, Makassar, South Sulawesi, 90245, Indonesia v1 First published: 23 Jul 2021, 10:645 Open Peer Review https://doi.org/10.12688/f1000research.53946.1 Latest published: 23 Jul 2021, 10:645 https://doi.org/10.12688/f1000research.53946.1 Reviewer Status AWAITING PEER REVIEW Any reports and responses or comments on the Abstract article can be found at the end of the article. Background: In Indonesia, malaria incidence is at a high rate despite maximum preventive efforts. Therefore, this study aims to determine the possibility of a Plasmodium reservoir among domestic animals in malaria-endemic areas. Methods: Animal blood was collected using EDTA tubes, then smeared and stained with Giemsa for Plasmodium microscopic identification. About 10 µl of blood was dropped on to a filter paper to capture Plasmodium DNA. Nested PCR was used for parasite molecular detection, while Plasmodium species were identified using the sequenced DNA. Results: A total of 208 and 62 animal blood samples were collected from Gaura village, West Sumba and Fakfak village, West Papua, Indonesia respectively. In total, 32 samples from Gaura contained P. falciparum or P. vivax, while the Plasmodium percentage in buffalo, horse, goat, and dogs were 20.7%, 14.3%, 5.8%, 16.7%, respectively. P. knowlesi was not found in any of the samples, and no other species were detected in 18 pig blood samples. Conclusion: Human Plasmodium existence among domestic animals in Indonesia partly explains the high prevalence and persistence of malaria in some endemic areas due to a reservoir host presence. Therefore, future studies need to ascertain the cause. Keywords Plasmodium falciparum, Plasmodium vivax, malaria, animals, host reservoir, PCR. Page 1 of 11 F1000Research 2021, 10:645 Last updated: 27 JUL 2021 Corresponding author: Sitti Wahyuni ([email protected]) Author roles: Munirah M: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Resources, Writing – Original Draft Preparation, Writing – Review & Editing; Wahyuni S: Conceptualization, Data Curation, Funding Acquisition, Methodology, Project Administration, Writing – Original Draft Preparation, Writing – Review & Editing; Wahid I: Data Curation, Formal Analysis, Methodology, Supervision, Writing – Original Draft Preparation; Hamid F: Formal Analysis, Supervision, Visualization, Writing – Original Draft Preparation Competing interests: No competing interests were disclosed. Grant information: The research was supported by Directorate of Research and Community Service. Directorate General of Research and Development Strengthening Ministry of Research, Technology and Higher Education, according to the Research Contract Number: 149 / SP2H / LT / DPRM / 2018 August 21, 2018. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2021 Munirah M et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite this article: Munirah M, Wahyuni S, Wahid I and Hamid F. The discovery of human Plasmodium among domestic animals in West Sumba and Fakfak, Indonesia [version 1; peer review: awaiting peer review] F1000Research 2021, 10:645 https://doi.org/10.12688/f1000research.53946.1 First published: 23 Jul 2021, 10:645 https://doi.org/10.12688/f1000research.53946.1 Page 2 of 11 F1000Research 2021, 10:645 Last updated: 27 JUL 2021 Introduction Malaria is transmitted by the Plasmodium vector Anopheles mosquitoes. Four Plasmodium types, namely P. falciparum, P. vivax, P. ovale, and P. malariae cause pathologic conditions in humans. Recently in Southeast Asia, P. knowlesi infection cases have also been reported.1–3 Before molecular diagnostics development, only humans were assumed to be the primary host for Plasmodium. However, studies in the last two decades on Plasmodium reported that the parasites originated from animals. Further stating that P. falciparum originated in the gorilla4 and chimpanzee,5,6 P. vivax was from African apes,7 P. malariae was from chimpanzees6 and P. knowlesi was from monkeys,8,9 while P. ovale in humans and chimpanzees are genetically identical.10 The factors hypothesized to explain this situation include primate’s habitat loss and human’s aggressiveness in exploring forest.11 A study from South Kalimantan reported the contribution of forest workers to malaria incidence.12 East Nusa Tenggara and West Papua are known as malaria-endemic areas in Indonesia as their annual parasite incidence (API) in 2015 was 31.29% and 7.04%, respectively,13 while in 2018, according to the health office in both the districts, the API rate in Fakfak, West Papua and East Nusa Tenggara, West Sumba was 4.85% and 12.9%, respectively.(unpublished data) Due to this situation, we aimed to explore the presence of human Plasmodium among domestic animals that are a potential reservoir host. Methods Study area and population This study was conducted in October 2018 in Gaura village, West Sumba Regency, an area 29.96 km2 in size inhabited by 9,584 people, and Fakfak, West Papua Province, in August 2019 with an area of 11,036 km2 inhabited by 84,692 people (Figure 1). The residents’ main occupation is farming, while livestock such as goats, horses, cows, pigs, and buffalos are commonly found in their enclosures located around the owner’s residence. Furthermore, they also own pets such as dogs and cats. Sample collection Sampling was carried out by the veterinarian and staff from West Sumba and Fakfak Animal Husbandry Office. The buffaloes, goats, pigs, and horses’ blood samples were collected in 5 ml EDTA tubes from the jugular vein located in the ventrolateral area of the neck using vacutainer needles, size 16–18. Meanwhile, the dog’s blood was drawn from the cephalic antebrachial vein in the leg using a size 21 vacutainer needle. By using a micropipette, approximately 10 ul of EDTA blood was dropped onto a microscope slide, then smeared and stained with Giemsa (MERCK Millipore, Germany) for Plasmodium microscopic identification, while the remaining was dropped onto a filter paper (Whatman CAT No. 1442-090) until it absorbed to about 1.5 cm in diameter. The dry filter paper was put on a sterile plastic clip and stored at room temperature for a maximum of 10 days. DNA extraction A dried blood spot (DBS) isolation kit for DNA extraction on filter paper (Cat. no. 36000) from Norgen Biotec was used. A6x3mmpiece of blood-stained filter paper was put into a 1.5 ml tube containing 100 μl of digestion buffer B. It was vortexed and incubated at 85°C. Afterwards, 20 μl of proteinase K and 300 μl of lysis buffer B were added to the tube and then vortexed before incubation at 56°C for 10 minutes. About 250 μl of 95% ethanol was added to the tube and then vortexed, while the DNA content was washed by adding 500 μl of WN wash solution and centrifugated for one minute at 8,000 rpm. Washing was carried out again using 500 μl of WN wash solution and centrifugated at 14,000 rpm. For DNA elution, 90 μl of elution buffer B was put into the tube and centrifuged at 8,000 rpm for one minute, and the purified DNA was stored at -20°C. DNA amplification and electrophoresis DNA amplification of nested PCR and qPCR were performed as directed by Tiangen Biotech (Beijing). Plasmodium DNA amplification was carried out using the nested PCR method with a 2Â Tag Plus PCR mix enzyme (Tiangen). The final volume of 12.5 μl contained 6.25 μl enzyme, 2.25 μl ddH2O, 1 μl forward primers, 1 μl reverse primers, and 2 μl DNA sample. For sequencing, the PCR mixture’s volume was doubled, with the final volume being 25 μl, while the primer sequences of P. falciparum, P. vivax14 and P. knowlesi15 can be seen in Table 1. The nested one DNA amplification temperature was set at 94°C denaturation (one minute), 55°C annealing (one minute) and 72°C extension (one minute) for 35 cycles. For nested two, denaturation was carried out at 94°C (30 seconds) and extension was at 72°C (30 seconds) in 35 cycles. There was a difference in the annealing temperature for each species in nested two, namely 55°C (one minute) for PCR multiplex P. falciparum and P. vivax, but 56°C (one minute) for P. knowlesi. Nested one products were used as templates for nested two and both were run on agarose gel 1.5% and 2%, Page 3 of 11 F1000Research 2021, 10:645 Last updated: 27 JUL 2021 Figure 1. Study area: Map of Gaura Village, West Lamboya, West Sumba, Indonesia and Fakfak Regency, West Papua, Indonesia. respectively, while qPCR was analysed using agarose gel 1.5% for electrophoresis. Molecular work was not performed for P. ovale and P. malariae due to difficulties in finding the positive control, and according to the local health office these species have never been reported from Sumba and Fakfak. Considering the possibility of contamination, DNA was re-extracted from blood from the same filter paper. PCR was performed using the primers, rPF1 and rPF2, as well as rPV1 and rPV216 to detect P. falciparum and P. vivax, respectively. The same extraction and amplification method were used as described above.